The broad long-term objectives for the research described in this proposal are aimed at a determination of structure-function relationships in catalytically active proteins and enzymes. The primary focus of this proposal will be directed towards the enzyme phosphotriesterase and related members of the amidohydrolase superfamily. This group of proteins catalyzes the nucleophilic attack of an activated water molecule on a diverse set of substrates. Phosphotriesterase catalyzes the detoxification of organophosphorus nerve agents while other members of this superfamily catalyze the hydrolytic cleavage of a carbonyl group attached to oxygen, nitrogen or chlorine. Since highly toxic organophosphate molecules are of rather recent environmental origin, the elucidation of the catalytic functions within phosphotriesterase, and the related proteins of the amidohydrolase superfamily, will provide unique insights into the mechanisms for the evolution and development of novel enzymatic activities. To define and articulate the factors that contribute to the determination of substrate specificity, random and site-directed variants of phosphotriesterase, and selected members of the amidohydrolase superfamily, will be constructed. The elucidation of the chemical reaction mechanism will be addressed by the construction of metal-substituted variants, site-directed mutants, and substrate analogs. The kinetic properties of the mechanistic probes will be utilized in the formulation of a self-consistent description of the catalytic events within the active sites. To more fully understand the factors that direct the evolution of new catalytic activities, the mechanisms of action and the three-dimensional structures of prominent members of the amidohydrolase superfamily will be determined. These studies will provide a historical window for how new catalytic activities can evolve from pre-existing enzymes. These investigations will complement concurrent efforts to direct the evolution of new and enhanced activities from present-day proteins.